PROJECT SUMMARY Glucose is one of the most highly regulated metabolites for nearly all organisms. In humans, misregulation of glucose metabolism plays a role in many diseases, from systemic metabolic syndrome to cancerous cell growth. This proposal focuses on the glucose transporters (GLUTs) in the solute carrier family 2 (SLC2), which includes the well-known transporters GLUT1, GLUT2, GLUT3, and GLUT4. These transporters facilitate the movement of glucose across the cell membrane following concentration gradients in an energy-independent manner. Since GLUTs serve as gates between the cell and the extracellular environment, their activity must be tightly controlled by both signaling events and metabolite levels. Despite their key roles in the major metabolic tissues, very little is known about GLUT protein-level regulation. Insulin regulation of GLUT4 is the notable exception, but even in this case, many mechanistic details (such as insulin-dependent inhibition of GLUT4 endocytosis) remain unknown. We discovered that an ?-arrestin protein, TXNIP (thioredoxin interacting protein), acts as an adaptor between GLUT1 and the clathrin-dependent endocytosis machinery, facilitating GLUT1 endocytosis. The interaction between TXNIP and GLUT1 is signal-dependent: phosphorylation of TXNIP by AMPK (AMP-activated kinase) dissociates TXNIP from GLUT1, preventing GLUT1 endocytosis, thus leading to a rapid increase in glucose influx. Given the sequence and structural homology among the GLUTs, together with signal-induced post- translational modifications found on the TXNIP protein, we aim to show that TXNIP is a universal regulatory node evolved for modulating acute glucose flux response. Specific Aim 1 will analyze the regulation of TXNIP on GLUTs, using GLUT1 as the prototype and developing a detailed map of its interaction through various biochemical assays. The information we learn about GLUT1 will be tested on other GLUTs, particularly GLUT4, to verify the role of TXNIP as a common adaptor for all GLUTs. In this Aim, we will also examine the role of lipids in TXNIP-GLUT interactions to build a foundation for future investigation of GLUT protein trafficking. Specific Aim 2 will focus on the upstream regulation of TXNIP by cell signaling events, particularly growth factor stimulation. We intend to identify upstream kinases and their phosphorylation sites on TXNIP; we also plan to test the effect of this regulation in vivo, using insulin regulation of glucose uptake in muscle and adipose tissue as the model. Muscle- and adipose-specific TXNIP knock-out mice will be generated to examine their metabolic profile. Combined understanding of the upstream and downstream regulatory mechanisms of TXNIP should provide greater insight into how glucose metabolism can be misregulated in human diseases.